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Antisense inhibition of ETs-2 expression

a technology of antisense and ets2, which is applied in the field of antisense compounds, can solve the problems that the target nucleic acid interferes with the normal function of the nucleic acid, and achieve the effect of increasing the viscosity of the external phase and preventing the deterioration of the formulation

Inactive Publication Date: 2000-04-25
IONIS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The specificity and sensitivity of aiitisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease status in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhan(ed affinity for nucleic acid target and increased stability in the presence of nucleases.
The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosirg schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vetry depending on the relative potency of individual oligonucle.otides, and can generally be estimated based on EC.sub.50 s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weigjht, and may be given once or more daily, weekly, monthly or ye(arly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

Problems solved by technology

The specific hybridization of an olicaomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2'-alkoxy amidites

2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2'-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2'-alkoxy amidites, the standard cycle for unmodified oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2'-deoxycytidine (5-Me-C) nucleotides were synthesized according to published methods [Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

2'-Fluoro amnidites

2'-Fluorodeoxyadenosine amidites

2'-fluoro oligonucleotides were synthe...

example 2

Oligonucleotide synthesis

Unsubstituted and substituted phosphodiester (P.dbd.O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P.dbd.S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetc,nitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55.degree. C. (18 h), the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5M NaCl solution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U...

example 3

Oligonucleoside Synthesis

Methylenemethylimino linked oligonicleosides, also identified as MMI linked oligonucleosides, methylenedimethyl-hydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecairbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P.dbd.O or P.dbd.S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

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Abstract

Antisense compounds, compositions and methods are provided for modulating the expression of Ets-2. The compositions comprise antisense com ounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Ets-2. Methods of using these compounds for modulation of Ets-2 expression and for treatment of diseases associated with expression of Ets-2 are provided.

Description

The present invention provides compositions and methods for modulating the expression of Ets-2. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human Ets-2. Such oligonucleotides have been shown to modulate the expression of Ets-2.The Ets-domain family of transcription factors is comprised of several proteins that are inviolved in controlling key cellular events such as proliferation, differentiation, and development. Their activity is often regulated by signal transduction pathways involving MAP kinases and, in regard to disease, the deregulation or mutation of Ets proteins is found primarily in cancers.While the Ets family is very diverse in protein sequence, all of the family members share a DNA binding domain known as the Ets-domain, so called because it resembles the protein product of the v-ets oncogene of the E26 avian erythroblastosis virus. Through this motif, the Ets family p...

Claims

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Application Information

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IPC IPC(8): C12N15/11A61K38/00C12N15/113
CPCC12N15/1135A61K38/00C12N2310/315C12N2310/321C12N2310/334C12N2310/346C12N2310/341C12N2310/3525Y02P20/582
Inventor BAKER, BRENDA F.COWSERT, LEX M.
Owner IONIS PHARMA INC